Use of branched-chain fatty acids and derivatives thereof for the treatment of pain

ABSTRACT

The present invention relates to compounds and pharmaceutical compositions useful for treating pain. The invention also relates to methods of treating or alleviating pain in mammals, comprising administering to a mammal suffering from pain, a pain-alleviating amount of a compound of the general formula (I) or (II).

This application is a national phase application (35 U.S.C. § 371) ofInternational Application No. PCT/IL02/00502 filed Jun. 24, 2002, whichis a continuation-in-part of U.S. patent application Ser. No.09/888,958, filed Jun. 25, 2001, now U.S. Pat. No. 6,518,311, which is acontinuation-in-part of U.S. patent application Ser. No. 09/462,533,filed Apr. 11, 2000, now U.S. Pat. No. 6,251,946, which is a nationalphase of International Application No. PCT/IL98/00316 filed on Jul. 7,1998.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment of pain. Moreparticularly, the invention relates to the use of certain branched-chainfatty acids (BFAs) and derivatives thereof in the treatment of pain inmammals and in particular in humans.

BACKGROUND OF THE INVENTION

Pain is a complex sensation and is the most common symptom of disease.Pain which is classified as somatogenic may be nociceptive pain which isdue to a noxious stimulus (chemical, thermal, mechanical etc.) thatactivates pain receptors, or neuropathic pain which results fromdysfunction of the central or peripheral nervous system and is oftenpoorly localized.

Pain may also be classified as being acute or chronic in nature. Acutepain is usually a result of injury (e.g. trauma or disease), it lasts ashort time and is typically resolves as the injured tissue heals or soonafter. Chronic pain is usually defined broadly and arbitrarily as a painpersisting for over one month beyond the resolution of an acute tissueinjury, pain persisting or recurring for more than 3 months, or painassociated with tissue injury that is continued or progressed [The MerckManual, 1999].

Several pain syndromes are difficult to classify according to thesecriteria. These include, for example, chronic headache and continuousacute pain produced by the invasion of body tissues in malignantdiseases.

Neuropathic pain is a common type of pain which can develop after injuryto the nervous system and is usually a chronic pain. Neuropathic painmay result by trauma, central nervous system (CNS) pathology (e.g.stroke, spinal cord injury), by diseases such as diabetes, herpeszoster, HIV infections or late stage of cancer, or by chemical injury.It may also develop after amputation.

The long-lasting neural mechanisms associated with chronic pain differfrom those observed in acute pain [Loeser and Melzack (1999) Lancet 353:1607-9]. Accordingly, also the pharmaceutical agents used to treat thevarious pain syndromes are different. It is well known that drugs thatare useful against acute pain may be ineffective against chronic painand drugs active, for example, against neuropathic pain may not beeffective analgesics in other kinds of pain.

A variety of analgesic agents have been demonstrated as useful in thetreatment of pain symptoms. Yet so far, the majority of availableanalgesics possess undesirable side effects.

Opioids are substances that act as agonists of opioid receptors in theCNS, and are considered as the most potent analgesic agents. Agents suchas morphine and related opioid compounds, are often required for reliefof severe pain. However, these narcotic drugs have the severe drawbackof leading to dependence and addiction. In addition, patients treatedwith opioids tend to develop tolerance to the drug, which leads toincreasing dosage of the drug needed for exerting the analgesic effectand to subsequent withdrawal symptoms. Further side effects associatedwith opioid drugs include nausea, sedation and respiratory depression.

Nonopioid analgesics, e.g. cyclooxygenase inhibitors such asacetaminophen (=paracetamol) and nonsteroidal anti-inflammatory drugs(NSAIDs) are often effective for treatment of mild to moderate pain.Anti-inflammatory agents of the NSAID class such as acetylsalicylic acid(aspirin), indomethacin, diclofenac and benzydamine have been used asanalgesics in pain associated with trauma and inflammation.Nevertheless, clinical trials are still inconclusive. Common sideeffects of the NSAID class of drugs include: gastrointestinal irritationand ulceration, blockade of platelet aggregation, renal dysfunction andhepatic damage.

Another major class of analgesics is the local anaesthetics that blocksodium channels. Compounds of this class, e.g. lidocaine, when topicallyapplied to the spine, have been found effective for control of painafter surgery or trauma, but require expertise and infrastructure toadminister and monitor properly. Systemic infusion of lidocaine canreduce acute pain, but requires continuous monitoring so thatresuscitation from seizures or apnea can be performed immediately.

N-methyl-D-aspartate (NMDA) receptor antagonists are useful in treatingneuropathic pain. It has been found that several sites on the NMDAreceptor complex, activated by the excitatory amino acid glutamate, areanalgesic targets. For example, Ketamine, which blocks the open calciumchannel within this complex, has been suggested for use preoperativelyor in neuropathic pain. Clinical studies confirm ketamine's merit as ananalgesic or co-analgesic (e.g. with morphine). Nevertheless,psychotomimetic and other side effects such as salivation or cardiacstimulation restrict the applicability of standard doses of ketamine[Martindale: The Extra Pharmacopeia. 31^(st) Edition. London:Pharmaceutical Press, (Editor Reynolds) 1996, pg. 1258-9].

Antidepressants, e.g. the tricyclic antidepressants amitriptyline andimipramine, and the serotonin re-uptake inhibitor paroxetine, have alsobeen proven beneficial as analgesics. Tricyclic antidepressants can behelpful in several chronic pain states, especially in patients with headpain (including headache), central pain, and neuropathic pain. However,these drugs have the potential for adverse side effects, includinganticholinergic effects and life-threatening cardiovascular effects.

Anticonvulsants have also been found to have useful analgesic effects.Gabapentin, has shown promise for the treatment of chronic pain[Rowbotham et al. (1998) JAMA 280: 1837-42] and carbamazepine andphenytoin, can be effective in the treatment of a range of neuropathicpain states. In particular, it has been found that trigeminal neuralgiaresponds well to carbamazepine [Green and Selman (1991) Headache 31:588-592]. Sodium valproate has been reported as being effective in theprophylactic treatment of migraine [Hering & Kuritzky (1992) Cephalalgia12: 81-84]. However, sodium valproate was found ineffective in theplacebo controlled study in treating postoperative pain, which is anacute pain [Martin et al. (1988) Ann Fr Anesth Reanim 7:387-92]. Someanalogs of valproic acid that have been tested as potentialanticonvulsant drugs were found sedative or had toxic effects [Keane etal. (1983) Neuropharmacology 22: 875-879].

Serious side effects that have been reported with anticonvulsant drugs,including deaths from hematological reactions, impaired mental and motorfunction, may limit clinical use, particularly in elderly people[Martindale: The Extra Pharmacopeia. 31^(st) Edition. London:Pharmaceutical Press, (Editor Reynolds) 1996, pgs. 367-381]. Moreover,the results of several clinical trials with anticonvulsant drugs weredisappointing and show conflicting results [McQuay et al. (1995) BMJ311:1047-1052]. In particular, there is no evidence that anticonvulsantsare effective for acute pain [Wiffen et al. (2000) The Cochrane Library,Issue 4, Oxford: Update Software].

Overall, it seems that the therapeutic effects of many of the existinganalgesic agents are controversial and often inadequate. In addition,most of the currently available analgesic medicaments suffer fromserious drawbacks which limit their use.

Clearly, there is an unmet clinical need for novel substances foreffective treatment of various forms of pain, including acute andneuropathic pain.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide methods for the treatmentand/or prophylaxis of pain in mammals, in particular acute andneuropathic pain in humans.

Thus the present invention relates, in one aspect, to methods oftreating or alleviating pain in mammals, comprising administering to amammal suffering from pain, a pain-alleviating amount of a compound ofthe general formula (I) or (II) or pharmaceutically acceptable saltsthereof.

The useful compounds according to the invention are of the generalformula (I):

and pharmaceutically acceptable salts thereof, wherein:

-   R₁ is a saturated or unsaturated chain of 1-18 carbons in length;    and-   R₂ is a saturated or unsaturated chain of 1-18 carbons in length,    with the proviso that R₁ and R₂ are not both propyl;

and compounds of the general formula (II):

and pharmaceutically acceptable salts thereof, wherein:

-   R₁ is a saturated or unsaturated chain of 1-18 carbons in length;-   R₂ is a saturated or unsaturated chain of 1-18 carbons in length;    and-   A is selected from the group consisting of PO₄—X, COOL and CONR′—R″,    wherein X is a hydrogen or choline, L is a lipid moiety selected    from the group consisting of glycerol, C₃₋₂₀ fatty acid    monoglycerides, C₃₋₂₀ fatty acid diglycerides, hydroxy-C₂₋₆-alkyl    esters of C₃₋₂₀ fatty acids, hydroxy-C₂₋₆-alkyl esters of    lysophosphatidic acids, lyso plasmalogens, lysophospholipids,    lysophophatidic acid amides, glycerophosphoric acids, sphingolipids,    lysophosphatidylethanolamine, and N-mono-(C₁₋₄)alkyl and    N,N-di-(C₁₋₄)alkyl and quaternary derivatives of the amines thereof;    and R′ and R″ are each independently selected from the group    consisting of hydrogen and a lower alkyl group comprising 1-5 carbon    atoms.

The methods encompassed by the invention, include methods for treatmentand/or prophylaxis of pain in mammals, and in particular in humans. Saidpain may be acute, chronic or neuropathic pain.

According to one preferred embodiment, the acute pain is selected from,but not limited to, post-operative pain, labor pain, toothache, paininduced by burns, muscle pain and pain accompanying myocardialinfraction.

According to another preferred embodiment of the invention, theneuropathic pain is selected from, but not limited to, post-herpeticneuralgia, diabetic neuropathy, trigeminal neuralgia and pain due toneural ischemic injuries, neural compression, demyelination andamputation.

According to yet another preferred embodiment, the treated pain isassociated with a pathological condition or disease state which may beselected from, but not being limited to, the group consisting of stroke,spinal cord injury and peripheral nerve injury, inflammation condition,cancer, trigeminal neuralgia, arthritis, sickle cell disease,hemophilia, diabetes, herpes zoster, HIV infections and headacheincluding cluster headache.

In currently preferred embodiments, the useful compounds are selectedfrom the group consisting of:

-   2-Pentylheptanoic acid [M(5,5)],-   2-Propyldodecanoic acid [M(3,10)],-   2-Propylnonanoic acid [M(3,7)],-   2-Heptylnonanoic acid [M(7,7)],-   1-O-stearoyl-2-propylnonayl-sn-glycero-3-phosphocholine [DP-M(3,7)],-   1-O-stearoyl-2-pentylheptonayl-sn-glycero-3-phosphocholine    [DP-M(5,5)],-   1-O-stearoyl-2-heptylnonayl-sn-glycero-3-phosphocholine [DP-M(7,7)],-   1-O-stearoyl-2-propyldodecanoyl-sn-glycero-3-phosphocholine    [DP-M(3,10)],-   8-Pentadecanyl Phosphate [(7,7)-PO₄], and-   8-Pentadecanyl phosphocholine [(7,7)-P-choline].

In another aspect, the present invention relates to compounds of theformula

wherein:

-   R₁ is a saturated or unsaturated chain of 6-18 carbons in length;-   R₂ is a saturated or unsaturated chain of 1-18 carbons in length and-   A is selected from the group consisting of PO₄—X, COOL and CONR′—R″,    wherein X is a hydrogen or choline, L is a lipid moiety selected    from the group consisting of glycerol, C₃₋₂₀ fatty acid    monoglycerides, C₃₋₂₀ fatty acid diglycerides, hydroxy-C₂₋₆-alkyl    esters of C₃₋₂₀ fatty acids, hydroxy-C₂₋₆-alkyl esters of    lysophosphatidic acids, lyso plasmalogens, lysophospholipids,    lysophophatidic acid amides, glycerophosphoric acids, sphingolipids,    lysophosphatidylethanolamine, and N-mono-(C₁₋₄)alkyl and    N,N-di-(C₁₋₄)alkyl and quaternary derivatives of the amines thereof;    and R′ and R″ are each independently selected from the group    consisting of hydrogen and a lower alkyl group comprising 1-5 carbon    atoms;-   and pharmaceutically acceptable salts thereof.

In one preferred embodiment, the compounds of the invention are thoseincluding a phospholipid moiety at position A. More preferred arecompounds having a lyso-phosphocholine moiety at position A. Currently,the most preferred useful phospholipid compound in accordance with theinvention is 1-O-stearoyl-2-pentylheptonayl-sn-glycero-3-phosphocholine[DP-M(5,5)].

In still another aspect, the present invention relates to pharmaceuticalcompositions for treating pain, comprising a pain-alleviating amount ofa compound of the formula mentioned above and at least onepharmaceutically acceptable carrier.

Further objects of the present invention will become apparent to thoseskilled in the art upon further review of the following disclosure,including the detailed descriptions of specific embodiments of theinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts analgesic effects of 25 mg/kg (grey bars), 50 mg/kg(striped bars) and 100 mg/kg (dark bars) of DP-M(5,5) as tested in miceusing the radiated heat ‘tail flick’ assay. The calculated percentageincrease in the withdrawal latency in comparison to the initialwithdrawal time (at t=0) is shown at different time points followingi.p. administration of the drug. The white bars represent the controlgroup of-animals treated with vehicle only.

FIG. 2 depicts the analgesic effect of 8-Pentadecanyl phosphocholine[(7,7)-P-choline] assayed in formalin induced pain test in mice. Thedrug, (7,7)-P-choline 150 mg/kg (squares), was orally administrated onehour before injection of formalin. Naive animals served as control group(circles). Statistically significant effect (p<0.05) is indicated byasterisks (*).

FIG. 3 depicts the effect of 8-Pentadecanyl phosphocholine[(7,7)-P-choline] as tested in an-assay for mechanical allodynia.Withdrawal threshold units represent the logarithm of 10 times the forcein milligrams required to bow the von Frey filament. Rats, at day 15post-CCI operation, were s.c. injected with either 150 mg/kg(7,7)-P-choline (squares) or vehicle only (circles).

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the invention to provide new analgesic compositionssuitable for the treatment of pain in mammals, and in particular for thetreatment of acute and neuropathic pain in humans.

A desirable analgesic agent is a compound capable of exerting itsfavorable analgesic effect by effectively reducing or alleviating painsensations, while having reduced or no side effects. In addition, along-lasting analgesic effect of the drug may also be beneficial in somecases of pain treatment.

According to the invention, compounds of the general formulas I and IIand pharmaceutically acceptable salts thereof are useful for treatmentof pain, that includes acute and chronic pain.

Unexpectedly, the inventors were now able to show that certainbranched-chain fatty acids and derivatives thereof have marked effectagainst acute pain and in chronic and neuropathic pain, as isdemonstrated by studies using acceptable animal model systems. Moreover,it has now been found that, surprisingly, some members of the family ofcompounds of the general formula I, which were not shown to be effectiveanticonvulsants in assays previously described in the parent applicationU.S. Ser. No. 09/462,533 (International publication WO 99/02485), havesignificant analgesic activity.

In the specification, the branched-chain fatty acid compounds of thegeneral formula I, or pharmaceutical acceptable salts thereof, will becollectively referred to as “BFAs”. Specific BFAs will be referred to,hereinafter, according to the number of carbons in their particular R1and R2 alkyl chains. For example, valproic acid which has propyl groupsat both R1 and R2 positions, is defined as M(3,3). Similarly,2-propylnonanoic acid is defined as M(3,7), 2-heptylnonanoic acid isdefined as M(7,7) etc.

R1 and R2 alkyl chains of BFAs may be straight or branched chains,saturated or unsaturated chains having one or more double and/or triplebonds.

The salt forms of the branched fatty acids will be referred to by asuffix including the salt symbol. For example, the sodium salt form of2-propylnonanoic acid is defined as M(3,7)-Na.

Suitable salts of the compounds of the general formula I include anypharmaceutically acceptable counter ion. In certain preferredembodiments the counter ion is selected from, but not limited to, Na⁺,Li⁺, K⁺, NH₄ ⁺, Ca⁺⁺ and mixtures of these ions.

Derivatives of branched-chain fatty acids of the general formula II andpharmaceutically acceptable salts thereof are also useful analgesiccompounds in accordance with the invention. These compounds in which thehydrogen atom of the carboxyl group of the branched fatty acid isreplaced by a lipid moiety (ester derivatives of BFAs), or the hydroxylgroup of the carboxyl group is replaced by a phosphate or aphosphocholine (ether derivatives of BFAs) or by an amine group (amidederivatives of BFAs), will hereinafter be collectively referred to as“DP-BFAs”.

The BFAs and DP-BFAs compounds can be prepared essentially by theprocesses described in U.S. application Ser. No. 09/462,533, (thedisclosure of which is herein incorporated in its entirety byreference), or by similar or alternative processes as they are known inthe art.

As mentioned above, the branched-chain lipophilic molecules may be usedas free acids, their physiologically acceptable salts or mixturesthereof, esters, ethers and amides.

Currently preferred useful compounds in accordance with the inventioninclude the fatty acid residues 2-Pentylheptanoic acid [M(5,5)],2-Propyldodecanoic acid [M(3,10)], 2-Propylnonanoic acid [M(3,7)] and2-Heptylnonanoic acid [M(7,7)]. Both the free acid and salt forms of thecompounds are useful analgesics in accordance with the invention.

Other preferred useful compounds are derivatives of BFAs (=DP-BFAs)wherein the hydrogen atom of the carboxyl group of the BFA is replacedby a lipid moiety, preferably a polar lipid and more preferably aphospholipid. Thus, preferred embodiments of the invention encompass theuse of the following DP-BFA compounds:

-   1-O-stearoyl-2-propylnonayl-sn-glycero-3-phosphocholine [DP-M(3,7)],-   1-O-stearoyl-2-propyldodecanoyl-sn-glycero-3-phosphocholine    [DP-M(3,10)],-   1-O-stearoyl-2-pentylheptonayl-sn-glycero-3-phosphocholine    [DP-M(5,5)] and-   1-O-stearoyl-2-heptylnonayl-sn-glycero-3-phosphocholine [DP-M(7,7)].

Other preferred useful compounds are the DP-BFA phospho derivatives8-Pentadecanyl Phosphate [(7,7)-PO₄] and 8-Pentadecanyl phosphocholine[(7,7)-P-choline].

Of the preferred members of both Formula I and formula II, the currentlymost preferred compounds are 2-Pentylheptanoic acid [M(5,5)],1-O-stearoyl-2-pentylheptonayl-sn-glycero-3-phosphocholine [DP-M(5,5)]and 8-Pentadecanyl phosphocholine [(7,7)-P-choline].

It is important to note that when a DP-BFA molecule of the generalformula II includes a glycerol based moiety at position A, the branchedfatty moiety may be linked to the glycerol-based moiety at any one ofpositions sn-1, sn-2 or sn-3. Linkage at position sn-2 of aphospholipid, namely BFA linked to a lyso-phospholipid moiety, ispreferred in accordance with the invention.

The DP-BFAs may be active per se or as prodrugs, which may be cleaved byan enzymatic or non-enzymatic reaction, preferably at the target site.For example, it has been reported that increase of phospholipase A₂activity is associated with inflammation and neuropathic pain [Kawakami(1998) Clin Orthop. 351:241-51; Saal et al. (1990) Spine 15:674-8].Hence, BFAs which are bound, for example, to a phospholipid at the sn-2position, may be released by these enzymes whose activity is elevated atthe site of pain.

The preferred type of lipid moiety selected for the generation of aDP-BFA compound, may depend on the specific disorder or pathologyassociated with the pain and its location. For example, sphingolipidswhich are found especially in nervous tissue and cell membranes may bepreferred lipids in the case where the analgesic compound is to betargeted to the brain or other CNS tissues.

The DP-BFAs compounds, being amphiphilic in nature, may penetratebiological membranes and barriers, thus facilitating the transport ofthe drug into privileged tissues and organs. Moreover, the DP-BFA drugs,having the BFAs covalently linked to a lipophilic moiety, may exhibitfavorable therapeutic activity e.g. improved pharmacokinetic propertiesand potency. Indeed, it was shown by the inventors of the presentinvention that the DP-BFA derivatives are more advantageous drugs in atleast two aspects: (i) they are more potent as having increasedanalgesic effect on a molar basis in comparison to the correspondingbranched-chain fatty acids, and (ii) their effect generally lasts forlonger time, thus making them effective drugs for the treatment ofchronic pain. In addition at least some of the DP-BFAs were found to bemuch less sedating compared to their branched-chain fatty acidscounterparts, therefore DP-BFAs are expected to exhibit reduced sideeffects and toxicity.

Any suitable route of administration is encompassed by the inventionincluding, but not limited to, oral, intravenous, intramuscular,subcutaneous, inhalation, intranasal, topical, rectal, epidural,intrathecal, systemic transdermal application or other known routes.

In one preferred embodiment, the useful pharmaceutical compositions ofthe invention are administered orally or intravenously. In anotherpreferred embodiment the route of administration is by topical or localapplication. Preferred embodiments of the topical application includenasal and ocular applications.

The pharmaceutical compositions may be in a liquid, aerosol or soliddosage form, and may be formulated into any suitable formulationincluding, but not limited to, solutions, suspensions, micelles,emulsions, microemulsions, ointments, gels, patches, suppositories,capsules, tablets, and the like, as will be required for the appropriateroute of administration.

Compositions for oral administration may include, but are not limitedto, powders or granules, suspensions or solutions in water ornon-aqueous media, sachets, capsules or tablets. Thickeners, diluents,flavorings, dispersing aids, preservatives, emulsifiers or binders maybe desirable.

Formulations for topical administration may include, but are not limitedto, lotions, ointments, gels, creams, suppositories, drops, liquids,sprays and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be added. In the caseof topical application, the composition including a pain-alleviatingamount of a compound of the general formula I or II, is preferablyapplied on or in close proximity to the tissue associated with the pain.

Administration may follow the injury that induces the pain sensation, oralternatively may precede the insult or stimuli that are likely toprovoke pain. On early administration, medicaments including theanalgesic compounds of the general formula I or II, will be useful asprophylactic medicaments that may prevent, delay or alleviate theprogression of pain symptoms. For example, the useful compounds, inaccordance with the invention, may be administered to a patient beforegoing through a surgical procedure, a painful dental treatment, enteringthe final stages of labor contractions etc., thus resulting inprevention, amelioration or reduction in the levels of post-operative orother pain.

The dose ranges for the administration of the compositions of theinvention are those large enough to produce the desired analgesiceffect. The compounds of the general formula I or II may be employed ata daily dosage in the range of from about 0.01 gram to about 10 grams.

The dosing range of the medicament varies with the route ofadministration and the condition of the patient suffering from pain. Thedosage administered will also be dependent upon the age, sex, health,weight of the recipient, concurrent treatment, if any, frequency oftreatment, severity of the symptoms and the specific nature of the painto be treated. In addition, dosage should be modified according to thepatient response, as it is common in the practice that the tolerancethresholds of patients and their perception of painful sensation may bequite different. Dosage regimen and means of administration will bedetermined by the attending physician or other person skilled in theart.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES

The useful compounds in accordance with the invention, can be preparedby processes as described below in Examples 1 to 5, or by anyalternative processes as they are known in the art.

It should be appreciated that while, for the sake of clarity, thefollowing discussion and synthesis examples relate to straight-chainsaturated members of the BFAs and DP-BFAs families, also branched andunsaturated BFAs and DP-BFAs, are included within the useful compoundsin accordance with the disclosure of the present invention.

Various pharmaceutically acceptable salts of the BFA and DP-BFAmolecules could also be obtained including, but not limited to, sodium,potassium, ammonium and alkyl-ammonium salts and salts with divalentcounter-ions.

Example 1 Synthesis of 2-Pentylheptanoic acid [M(5,5)]

Anhydrous tetrahydrofuran (THF, 900 ml) and diisopropylamine (121.97 g,1.208 mol) were added to a dry argon flushed flask under an atmosphereof argon. n-Butyllithium in hexane (740 ml of 1.6 M, 1.184 mol) wasadded to the magnetically stirred solution at such a rate as to maintainthe temperature below 0° C. n-Heptanoic acid (80.81 g, 0.62 mol) wasthen added to the cold basic solution and again the temperature was keptbelow 0° C. After 15 min, HMPA (230 ml, 1.28 mol) was added to the milkywhite solution, which, after 5 min of staining at 5° C., becametransparent and light yellow. The solution was then stirred for anadditional 15 min at 5° C., and n-Pentyl bromide (89.32 g, 0.592 mol)was added at once at 0° C. The reaction temperature immediately rose to18-20° C. After 2 hours of additional stirring at room temperature, thereaction was processed in the following manner. Dilute hydrochloric acid(10%) was added at 0° C. until the mixture became acidic. The aqueouslayer was separated and extracted with petroleum ether (bp 30-60° C.).The combined organic layers were washed with dilute hydrochloric acidand H₂O. The organic layer was then dried and the solvent was removed.The residue was distilled. Yield, was 87 g (73%) of colorless oil, bp.85-90° (1 mm Hg).

TLC analysis: Silica gel 60 F254 on aluminum sheet. Eluent is a mixtureof pentane with ether (8:2 v/v).

2-Pentylheptanoic acid [M(5,5)]

Colorless oil, Bp. 85-90° C. (1 mm Hg). Yield=73%.

TLC analysis: One spot. R_(f.)=0.42.

NMR (CDCl3), δ (ppm): 0.85-0.95 (m, 6H), 1.28-1.33 (broad s, 12H),1.47-1.50 (m, 2H), 1.56 (m, 2H), 2.31 (m, 1H). MS (ES, negative. ionsmode)=198.99 (M−H)⁻.

The procedure for preparation of other BFAs is analogues to thesynthesis of the 2-Pentylheptanoic acid mentioned above. The analysiswas performed on TLC under the conditions as mentioned above.

2-Propyldodecanoic acid [M(3,10)]

Colorless oil, Bp. 128-138° C. (1 mm Hg). Yield=77%.

TLC analysis: One spot. R_(f.)=0.5.

NMR (CDCl₃), δ (ppm): 0.84(m, 6H), 1.2-1.33 (18H), 1.43 (m. 2H), 1.59(m.2H), 2.35 (m.1H). MS (ES, negative. ions mode)=241.13. (M−H)⁻

2-Propylnonanoic acid [M(3,7)]

Colorless oil, Bp. 128-135° C. (1 mm Hg). Yield=75%.

TLC analysis: One spot. R_(f.)=0.46.

NMR (CDCl₃), δ (ppm): 0.84(m, 6H), 1.2-1.33 (10H), 1.43 (m. 2H), 1.59(m.2H), 2.35 (m.1H). MS (ES, negative. ions mode)=199 (M−H)⁻

2-Heptylnonanoic acid [M(7,7)]

Colorless oil, Bp. 128-135° C. (1 mm Hg). Yield=75%.

TLC analysis: One spot. R_(f.)=0.46.

NMR (CDCl₃), δ (ppm): 0.84 (m, 6H), 1.2-1.33.(20H), 1.43 (m. 2H), 1.59(m.2H), 2.35 (m.1H). MS (ES, negative. ions mode)=254.4 (M−H)⁻.

2-Propyleicosanoic acid [M(3,18)]

Colorless oil, M.p. 55.6-57.4. Yield=75%.

TLC analysis: One spot. R_(f.=0.39.)

NMR(CDCl₃), δ (ppm): 0.84 (m, 6H), 1.2-1.33 (34H), 1.43 (m. 2H), 1.59(m.2H), 2.35 (m.1H). MS (ES, negative ion mode)=353.4 (M−H)⁻.

The synthesis of the salt forms of branched-chain fatty acids isexemplified below by the synthesis procedure for 2-heptylnonaoic acidsodium salt [M(7,7)-Na].

Anhydrous ethanol (120 ml) and 2-heptylnonaoic acid (10 gr, 0.039 mol)were added to a dry argon flushed flask under an atmosphere of argon.Sodium ethylate (69.92 ml of 0.53 M, 0.037 mol) was added to themagnetically stirred solution at room temperature. After 5-6 hours thesolvent was removed. The 2-heptylnonaoic acid sodium salt wasre-crystallized from acetone.

Sodium titration is 100%. NMR analysis is the same as for the2-heptylnonaoic acid.

Example 2 Synthesis of1-O-stearoyl-2-O-propylnonayl-sn-glycero-3-phosphocholine[DP-M(3,7)-ester]

The synthesis of1-O-stearoyl-2-O-propylnonayl-sn-glycero-3-phosphocholine [DP-M(3,7)] isa two-stage process. The first stage is the preparation of2-propylnanoic anhydride. The second stage includes binding of the BFAto the lipid moiety, in this case a lyso-lecithin, and isolation of thefinal product.

Stage I. Synthesis of 2-proylnonanoic Anhydride

In a round-bottom single-neck flask (250 ml), equipped with a reversecondenser (water cooling) and magnetic stirrer, 2-propylnonoic acid (100g, 0.5M), acetic anhydride (analytical, 400 ml, 1.06M) and pyridine(analytical, 44 ml, 0.5M) were introduced. This reaction mixture wasstirred by magnetic stirrer for 4 hours at 70° C. After that aceticanhydride was evaporated at a pressure of 20 mm Hg. Residue wasdistilled at 1 mm Hg and fraction which is boiling at 150-152° C. wascollected. This is 2-propylnonanoic anhydride. Yield was 85% (81.1 g).

Analyses: TLC is performed on plates of Silica gel 60 F254 (Merck).Eluent is chloroform (analytical). One spot was visible in UV spectra.Rf=0.89.

Elemental analysis: C₂₄H₄₆O₃. Calculated: C, 75.39%; H, 12.04%. Found:C, 75.25%; H, 11.95%.

STAGE II. Synthesis of1-O-STEAROYL-2-O-2-PROPYLNONAYL-sn-GLYCERO-3-PHOSPHOCHOLINE

Lyso-lecithin (1-stearoyl-sn-glycero-phosphocholine; 2 g, 3.82 mM),sodium salt of valproic acid (0.7 g, 4.2 mM) and 2-propyl-nonanoicanhydride (20 ml) were introduced under argon into a round-bottomsingle-neck flask (500 ml), equipped with a reverse condenser (watercooling) and a magnetic stirrer. The reaction mixture was heated in oilbath (80-100° C.) until disappearance of the lyso-lecithin (TLCmonitoring) in the solution (about 3 hours of heating). The unreactedvalproic anhydride was then distilled from the reaction flask by heating(110-120° C.) in vacuum (about 0.1 mm Hg). The residue was dissolved inchloroform and the precipitate of sodium salt of valproic acid wasseparated from the solution by centrifugation. The obtained solution wasconcentrated by heating in an evaporator. After cooling, the chloroformsolution of the reaction product was filtered on a chromatography columncomposed of Silica gel 60 (70-230 mesh). For purification of 1 gr. ofraw reaction product 30 gr. of Silica gel are used. A mixture ofchloroform, methanol and water (65:35:5 v/v) is used as an eluent.2-propylheptyl acetic acid and its unreacted anhydride appear with thefront of the eluent. The product is a white wax. After chromatography,purification of the product is performed by washing with n-pentane(three washes, each using a 20 ml portion). The obtained product wasdried in vacuum at 40° C. Yield was 60% (1.5 g) p TLC analysis: Silicagel 60 F254 on aluminum sheet. Eluent is chloroform (stabilized byamylene). One spot was visible in UV spectra. Rf=0.3.

Elemental analysis: C₃₈H₇₆O₈NP. Calculated: C, 64.62%; H, 10.77%; N,2.00%; P, 4.39%. Found: C, 64.00%; H, 10.9%; N, 2.21%; P, 4.4%. 1H NMR.(CDCL3), δ (ppm): 0.86-0.92 (m, 9H), 1.26 (broad s, 42H), 1.42-1.44 (m,2H), 1.53-1.61 (m, 4H), 2.24-2.34 (m, 3H), 3.38 (s, 9H), 3.81-4.45(broad m, 8H) and 5.20-5.28 (m,1H). 31P NMR (CDCL3), δ (ppm): −3.0(respectively H₃PO₄ in D₂O) (s)

Analysis data for some specific DP-BFA molecules are listed below.

1-O-stearoyl-2-propylnonayl-sn-glycero-3-phosphocholine [DP-M(3,7)]

Elemental analysis: M.2H₂O (calculated/found %): C=61.54/62.18±0.51,H=10.19/10.40±0.15, N=1.89/1.71±0.05, P=4.18/4.04. MS (ES, positive ionsmode)=706 (M+H)⁺. TLC: Silica gel 60 F254 on aluminum sheet. Eluent ismixture of chloroform-methanol-water (64:25:4). One spot. R_(f.)=0.68.NMR (CDCl₃), δ (ppm): 084(m, 9H), 1.25-1.37 (40H), 1.41 (m. 2H),1.49-1.59 (m.4H), 2.25-2.35(3H), 3.37 (9H), 3.38-4.48 (8H), 5.2 (m, H).

1-O-stearoyl-2-pentylheptonayl-sn-glycero-3-phosphocholine [DP-M(5,5)]

Elemental analysis: M.2H₂O (calculated/found %): C=61.54/61.36±0.05,H=10.79/9.91±0.34, N=1.81/1.75±00.4, P=4.18/3.65. MS (ES, positive ionsmode)=706 (M+H)⁺. TLC: Silica gel 60 F254 on aluminum sheet. Eluent ismixture of chloroform-methanol-water (64:25:4). One spot. R_(f.)=0.67.NMR (CDCl₃), δ (ppm): 084(m, 9H), 1.25-1.37 (40H), 1.41 (m. 2H),1.49-1.59 (m.4H), 2.25-2.35(3H), 3.37 (9H), 3.38-4.48 (8H), 5.2 (m, H).

1-O-stearoyl-2-heptylnonayl -sn-glycero-3-phosphocholine [DP-M(7,7)]

Elemental analysis: M.1H₂O (calculated/found %): C=64.70/64.05±0.08,H=11.04/11.21±0.13, N=1.80/1.81±00.3, P=4.0/3.02. MS (ES, positive ionsmode)=762 (M+H)⁺. TLC: Silica gel 60 F254 on aluminum sheet. Eluent ismixture of chloroform-methanol-water (64:25:4). One spot. R_(f.)=0.73.NMR (CDCl₃), δ (ppm): 084(m, 9H), 1.25-1.37 (48H), 1.41 (m. 2H),1.49-1.59 (m.4H), 2.27-2.35(3H), 3.22 (9H), 3.38-4.48 (8H), 5.2 (m, H).

1-O-stearoyl-2-propyldodecanoyl-sn-glycero-3-phosphocholine [DP-M(3,10)]

Elemental analysis: M.2H₂O (calculated/found %): C=63.56/63.52±0.12,H=11.24/10.33±0.03, N=1.80/1.65±00.4, P=4.00/4.28. MS (ES, positive ionsmode)=749 (M+H)⁺. TLC: Silica gel 60 F254 on aluminum sheet. Eluent ismixture of chloroform-methanol-water (64:25:4). One spot. R_(f.)=0.71NMR (CDCl₃), δ (ppm): 084(m, 9H), 1.28-1.35 (46H), 1.41 (m. 2H),1.49-1.59 (m.4H), 2.27-2.33(3H), 3.22 (9H), 3.38-4.48 (8H), 5.2 (m, H).

Example 3 Synthesis of 2-Propyloctadecanamide [DP-M(3,16)-amide]

The synthesis of the amide analogs of the branched chain fatty acid isexemplified below by the synthesis procedure for 2-propyloctadecanamide[DP-M(3,16)-amide].

The synthesis of the amide derivatives of BFAs compounds is a two-stepprocedure. The chloride derivative of the branched chain fatty acid isprepared at the first stage, followed by the addition of the amideitself at the second stage. The other DP-amides are prepared accordingto an analogous procedure to the synthesis of 2-propyloctadecanamidedescribed below.

Overall, the synthesis can be described according to the followingsynthetic pathway:

2-Propyloctadecanoic acid (200 mg, 0.61 mmol) was introduced into asingle neck round-bottom flask, equipped with a magnetic stirrer and areverse condenser. Three milliliters of SO₂Cl₂ were also placed in theflask. The reaction mixture was brought to reflux and left for one hour.The reaction mixture was then evaporated. Dry benzene (5 ml) was addedto the residue and then evaporated. This procedure was repeated twice.The resultant residue was dissolved in 5 ml of dry tetrahydrofuran andthen 0.2 ml of 0.5 M ammonia in dioxane was added to this solution. Thesuspension thus obtained was stirred with a magnetic stirrer for onehour and then evaporated. Petroleum ether was added to the residue andthe mixture was stirred. The obtained solid was filtered, and thenwashed twice with water and petroleum ether. The precipitate was driedin vacuum (10 mm Hg) for three hours at room temperature. The productwas a white powder. The yield was 80%.

2-Propyloctadecanamide [DP-M(3,16)-amide]

The product was a white powder with a yield of 80%.

TLC analyses: Silica gel 60 on aluminum sheet. Eluent was a mixture ofpetroleum ether with diethyl ether (3:7, v/v). Indicator was a spray of4-methoxybenzaldehyde (10 ml), abs. ethanol (200 ml), 98% sulfuric acid(10 ml), glacial acetic acid (2 ml). The chromatogram was sprayed withthis indicator, dried and then charred at 100-150° C. One spot wasobserved. R_(f)=0.2.

Elemental analysis: C₂₃H₄₇NO. Calculated: C, 78.19%; H, 13.31%; N,3.97%. Found: C, 78.09%; H, 13.11%; N, 3.77%. ¹H NMR. (CDCL₃), δ (ppm):0.84-0.93 (m, 6H), 1.24-1.41 (m, 34H), 1.52-1.60 (broad s, 4H),2.06-2.15 (m,1H) and 5.30-5.36 (d, 2H).

Analysis data for additional DP-BFA-amide molecules are listed below.

2-Propyl-nonamide [DP-M(3,7)-amide]

The product was a white powder with a yield of 75%.

TLC analyses: Silica gel 60 on aluminum sheet. Eluent was a mixture ofpetroleum ether with diethyl ether (3:7, v/v). Indicator was a spray of4-methoxybenzaldehyde (10 ml), abs. ethanol (200 ml), 98% sulfuric acid(10 ml), glacial acetic acid (2 ml). The chromatogram was sprayed withthis indicator, dried and then charred at 100-150° C. One spot wasobserved. R_(f)=0.2.

Elemental analysis: C₁₂H₂₅NO. Calculated: C, 72.36%; H, 12.56%; N,7.04%. Found: C, 72.50%; H, 12.81%; N, 7.22%. ¹H NMR. (CDCL₃), δ (ppm):0.85-0.94 (m, 6H), 1.22-1.42 (m, 14H), 1.51-1.60 (m, 2H), 2.06-2.18(m,1H) and 5.32-5.44 (d, 2H).

2-Propylhexadecanamide [DP-M(3,14)-amide]

The product was a white powder with a yield of 80%.

TLC analyses: Silica gel 60 on aluminum sheet. Eluent was a mixture ofpetroleum ether with diethyl ether (3:7, v/v). Indicator was a spray of4-methoxybenzaldehyde (10 ml), abs. ethanol (200 ml), 98% sulfuric acid(10 ml), glacial acetic acid (2 ml). The chromatogram was sprayed withthis indicator, dried and then charred at 100-150° C. One spot wasobserved. R_(f)=0.2.

Elemental analysis: C₂₁H₄₃NO. Calculated: C, 77.53%; H, 13.23%; N,4.30%. Found: C, 77.56%; H, 13.39%; N, 4.39%. ¹H NMR. (CDCL₃), δ (ppm):0.86-0.92 (m, 6H), 1.24-1.43 (m, 30H), 1.52-1.60 (m, 4H), 2.08-2.12(m,1H) and 5.28-5.36 (d, 2H).

Example 4 Synthesis of Alkyl Phosphates

Phosphates of the general formula RO—P(O)(OH)₂ were prepared. Rrepresents a branched-chain alkyl moiety of the R₁(R₂)—CH structurewhere R₁ indicates the number of carbons in the side alkyl chain and R₂indicates the number of carbons in the main alkyl chain.

The synthesis of RO—P(O)(OH)₂ molecules is a three-stage procedure. Inthe first stage the corresponding alcohol (R—OH) was prepared fromaldehyde and alkyl bromide using the Grignard reaction (Vogel's,“Textbook of practical organic chemistry”, Wiley, New York, pg. 531,(1996).)

In the second stage diphenyl phosphate ester was prepared from thealcohol and diphenyl phospochloridate:ROH+ClP(O)(OC₆H₅)₂+C₅H₅N→RO—P(O)(OC₆H₅)₂+C₅H₅N.HCl

In the third stage alkanyl dihydrogen phosphate was obtained byhydrogenation of the diphenyl ester.RO—P(O)(OC₆H₅)₂+2H₂→RO—P(O)(OH)₂+2C₆H₆

8-Pentadecanyl Diphenyl Phosphate

Diphenyl phosphorochloridate (4.0 g, 0.015 mole) was added slowly whileshaking to a solution of pentadecane-8-ol (2.28 g, 0.01 mole) in drypyridine (5 ml) at room temperature. The flask was stopped and set asidefor 48 hr.; then the contents were poured into ice-cold 1N hydrochloricacid (100 ml). The heavy oil, which separated was extracted with ether.The ethereal layer was washed with 1N hydrochloric acid (3 times), 5%sodium hydrogen carbonate (5 times), and water (5 times). After beingdried (MgSO₄), the ether was removed, and the residue was purified bycolumn chromatography (Petrol Ether (bp 30-60° C.): Ether, 10:1). Afterevaporation of a solvent 3.4 g of liquid was obtained. Yield 75%.

Synthesis of 8-Pentadecanyl Phosphate. [(7,7)-PO₄]

A suspension of platinum oxide (Adams catalyst) (0.32 g) in glacialacetic acid (20 ml) was shaken under hydrogen atmosphere untilabsorption ceased. The Adams catalyst was then washed well with 2Nhydrochloric acid, water, and finally glacial acetic acid, bydecantation. Solution of 8-Pentadecanyl diphenyl phosphate (3.0 g) inglacial acetic acid (40 ml) was added to the catalyst, and the solutionwas shaken under hydrogen until absorption ceased. The catalyst wasfiltered off and washed with chloroform. The solvents were removed fromthe filtrate in vacuum. The residue was crystallized from petroleumether (bp 30-60° C.) and dried at 65° C. 2.01 g of final product wasobtained. Yield 92%.

8-Pentadecanyl Phosphate [(7,7)-PO₄]

¹H-NMR (CD₃OD) δ: 0.83-0.88 (t., 6H), 1.25 (broad s., 20 H), 4.08 (broads., 1H) ³¹P-NMR (CD₃OD) δ: 3.69 s.

Example 5 Synthesis of Alkylphosphocholines

Phosphocholine compounds of the formula RO—PO⁻(O)—O—CH₂CH₂N⁺(CH₃)₃ wereprepared by the following procedure as described below for8-Pentadecanyl phosphocholine [(7,7)-P-Choline], while using thecorresponding R—OH alcohol in the initial step. R represents branchedchain alkyls of the R₁(R₂)—CH type.

Synthesis of 8-Pentadecanyl phosphocholine [(7,7)-P-choline]

To a cooled solution (0° C.) of pentadecane-8-ol (10.03 g, 0.044 mol)and triethylamine (10 ml, 0.075 mol) in dry ether (250 ml) was added2-chloro-2-oxo-1,3,2-dioxaphospholane (7 ml, 0.075 mol) in 200 ml of dryether. The mixture was stirred at room temperature for 2 hrs. Thecrystalline (C₂H₅)₃N.HCl that precipitated was filtered off, and thesolvent was removed in vacuum. The residue was dissolved in 500 mlsolution of trimethylamine (0.27M) in anhydrous acetonitrile andtransferred to a pressure bottle. The pressure bottle was kept for 48hrs in an oil bath at 60-65° C. The bottle was then cooled and opened.The solvent was removed, and the residue was purified by columnchromatography (CHCI₃:CH₃OH:H₂O, 1:9:1). The oil obtained afterevaporation of the solvent was lyophilized during 72 hrs at 65° C. 13 gof light yellow wax was obtained. Yield 75%.

8-Pentadecanyl phosphocholine [(7,7)-P-choline]

¹H-NMR (CD₃OD): δ 0.92 (t, 6H), 1.32 (s, 20H), 1.59 (m, 4H), 3.23 (S,9H), 3.63 (m, 2H), 4.26 (m, 3H). MS (FAB): m/z 394.35 (M+H)⁺. ³¹P-NMR(CD₃OD): δ: 0.61 s.

The following compounds were synthesized by a process analogous to theabove-described procedure.

4-Hexadecanyl phosphocholine (3,12-P-choline)

¹H-NMR (CD₃OD): δ 0.91 (t, 6H), 1.28 (s, 22H), 1.57 (m, 4H), 3.21 (S,9H), 3.62 (m, 2H), 4.25 (m, 3H). MS (FAB): m/z 408.68 (M+H)⁺.

4-Octadecanyl phosphocholine (3,14-P-choline)

¹H-NMR (CD₃OD); δ 0.9 (t, 6H), 1.28 (s, 26H), 1.57 (m, 4H), 3.21 (S,9H), 3.61 (m, 2H), 4.23 (m, 3H). MS (FAB): m/z 436.91 (M+H)⁺.

Example 6 Effects of BFAs and their Derivatives Tested in Tail-FlickAssay (a Model System for Acute Pain)

The analgesic effect of branched-chain fatty acids (BFAs) and theirDP-BFAs derivatives was tested by using the tail flick assay in mice.

The tail-flick test is a heat nociception test initially employed byD'Amour and Smith [D'Amour and Smith (1941) J. Pharmacol. Exp Ther.72:74-79] and is a widely used animal model system for quantitativemeasurements of acute pain threshold. This model uses radiated infrared(IR) heat source that is directed to the tail of a restrained mouse. Thethreshold of tolerance for heat is indicated by a time-meter, which isstopped instantaneously when the tail flicks. This time is defined as‘withdrawal latency’.

Male CD-1 mice weighing around 25-30 grams (4-8 animals per each dose ofthe tested compound) were used. The animals were put in clear plasticcages above an IR source (7371-Plantar™ Analgesia Instrument, UGOBasile), where the IR generator is placed directly underneath the tailof the mouse and the light beam is focused on the proximal third of thetail.

The withdrawal latency, namely the time interval from the starting ofthe infrared radiation until the animal feels pain and flicks its tailis determined. The initial withdrawal latency for each animal wasmeasured at t=0 and was determined as its baseline threshold.

In order to determine the analgesic effect of a tested drug, the drug(10 to 200 mg/kg body weight) was administered to mice either orally(p.o.) or by intraperitoneal (i.p.) or subcutaneous (s.c.) injection.Withdrawal latency was determined at different time pointspost-administration of the drug as indicated. Each reading was performed2-3 times and the mean-value was calculated. Withdrawal latencies of thetreated animals, expressed as percentage of the withdrawal latency ofthe control group of animals treated with vehicle alone, are shown inTable 1. The withdrawal latency at each time point post injection is theaverage of 4-8 animals per group. Withdrawal latency at t=0 for alltested animals was around 9 to 12 seconds. Paired T-test was used toassess significance in comparison to the baseline value (t=0). Morphinewas used as a positive control (2 animals per dose).

TABLE 1 Analgesic effects of BFAs and DP-BFAs measured in tail flickassay Withdrawal latency (% of vehicle) Time post-injection (min.) Dose15 45 75 105 135 285 345 Compound (mg/kg) (mmole/kg) min min min min minmin min I.P. Administration Morphine 1  97% 136% 207% 179%  153%* 142%** 10 139% 171% 284% 238%  190%*  151%** M(3,7) 150 750 157% 137%106% 131% M(3,10) 100 413 142% 155% 131% 156% 123% 200 826 186% 210%183% M(3,10)—Na 50 189 151% 138% 128% 123% 102% 100 378 181% 153% 121%143% 148% 200 756 158% 220% 216% 141% 175% M(5,5) 200 1000 161% 135%145% 158% M(5,5)—Na 50 225 115% 124% 117% 134% 100 450 137% 164% 164%193% M(7,7) 200 720  93% 124% 117% 149% 124% DP-M(3,7) 50 71 132% 123%119% 102% 112% 119% 114% 100 142 127% 156% 124% 114% 126% 127% 125%DP-M(3,10) 50 67 123% 119% 113% 123% 123% 129% 107% DP-M(5,5) 50 71 113%107% 118% 142% 152% 121% 124% 100 142 148% 143% 148% 162% 171% 155% 124%(7,7)-P-choline 100 254 235% 134% 177% 134% 141% 145% S.C.Administration (7,7)-P-choline 150 382 194% 217% 186% 175% 184% 175%(7,7)-P-choline 25 63.5 147% 143% 165% 169% 141% 130% P.O.Administration (7,7)-P-choline 50 127 149% 157% 145% 137% 127% 124%(7,7)-PO₄ 200 606  93%  94% 124% 133% 162% 129% *Time post-injection 165min. **Time post-injection 255 min.

As shown in Table 1, a statistically significant increase in withdrawallatency, indicating an increase in the animal pain threshold, can beseen with the different amounts of the BFAs and DP-BFAs molecules. Thelargest analgesic effects in this experiment were demonstrated with8-Pentadecanyl phosphocholine [(7,7)-P-choline],1-O-stearoyl-2-pentylheptonayl-sn-glycero-3-phosphocholine [DP-M(5,5)],2-Pentylheptanoic acid [M(5,5)] and 2-Propyldodecanoic acid [M(3,10)].

Generally, the DP-BFA derivatives were about 3-4 times more potent, on amolar basis, in comparison to their corresponding BFA molecules. Themost potent drug in this assay was (7,7)-P-choline.

It is important to note that the tested compounds exerted theiranalgesic effect in a dose dependent fashion (results for DP-M(5,5) areshown in FIG. 1). It should also be pointed out that duration of theanalgesia effect with most tested BFA compounds in this model system wasfor about 1.5-2 hours following dosing. With the lipid and phosphoderivatives, DP-BFAs, a significant effect was demonstrated for up to5-6 hours post injection. Prolonged effect of the drugs was alsodemonstrated with subcutaneously (s.c.) injected DP-BFA derivatives. Forexample, at 5-6 hours post s.c. injection, (7,7)-P-choline showed around80% increase in withdrawal latency in animals treated with 150 mg/kg ofthe drug.

Generally, the animals treated with the DP-BFA phosphatidylcholinederivatives were less sedative in comparison to the animals treated withthe corresponding BFAs compounds.

Conclusions: The results of the “tail-flick” study demonstrate that BFAsand DP-BFAs compounds are effective in reducing acute pain sensation.

Example 7 Effects of BFAs Tested in Writhing Assay (a Model forPeripheral Acute Pain)

The analgesic effects of BFAs were evaluated in an animal model systemfor peripheral acute pain, the writhing model system.

The writhing model represents a chemical nociceptive test, based on theinduction of a peritonitis-like condition in the animals by injectingirritant substances intraperitoneally (i.p.). The writhing test is asimple and reproducible assay, which is characterized by repeatedcontractions of the abdominal muscles accompanied by extension of thehindlimbs of the animal (Jaques, Arzneimittelforschung, 27, 1698-70,1977: Siegmund et al, Proc. Soc. Exp. Biol. 95:729-731, 1957).

Pain is induced in CD-1 mice by i.p. injection of acetic acid (0.6%, 10ml/kg in ddH₂O). The number of writhes (abdominal constriction followedby dorsiflexion and extension) occurring during a 15 min. time period isrecorded, starting 5 minutes after acetic acid administration.

Average of around 20 to 30 writhes were recorded during this period oftime in animals injected with the acetic acid only or animals treatedwith vehicle. The treated animals are subcutaneously (s.c.) injected, 30minutes prior to the injection of the acetic acid, with different dosesof the tested compounds in amounts ranging from 10 to 200 mg/kg bodyweight. Animals treated with vehicle only, serve as control.

Reduction in the number of writhes in response to acid that is injectedfollowing administration of the BFAs or DP-BFAs, demonstrates that thesecompounds may serve as effective analgesics useful for treatment ofacute pain.

Example 8 Effects of DP-BFAs on Formalin-Induced Pain in Mice

Formalin induced pain is an easy and reliable test in animals [Takahashiet al. (1984) Jpn. J. Oral Biol. 26:543-548]. Pain can be induced byformalin injection in a large number of species, including rodents, andis biphasic in nature. An early acute nociceptive phase (0 to 5 minpost-injection) is followed by a second phase that resembles neuropathicpain (around 15 to 60 min). The interphase period (quiescent phase),when the animals demonstrate the least expression of pain, is usuallyaround 5 to 15 min following the noxious stimulus.

Evaluation of pain is based on the observed behavior of the animalsafter the formalin injection. Many features of behavior are expressed,amongst which are licking of the paw, flinching, lifting the leg off theground, limping or general observed irritation in the injected foot.

Experimental Design

Formalin stock (Frutarom Ltd., Israel; cat. # 5551830; 3.4-3.8%formaldehyde content) was diluted into 2.5% formalin solution in saline.25 μl of the 2.5% formalin solution was injected into the plantarsurface of CD-1 mice foot using a syringe with a 29G needle. Theanimals' reaction, apparent as licking and lifting the paw off theground, is noticed immediately after injection. Each of the treatedanimals and control naive animals injected only with formalin wasmonitored for a period of 40 min, through 5 minutes time segments (4-6animals in a group).

Evaluation of pain was performed by recording the time the animal spentlicking its paw. A computerized program was used for timing the totalnumber of seconds each animal spent licking its paw during the presetperiod of 5 min. This step was repeated for up to 40 min (8 intervals of5 min).

The results recorded from such an experiment where 150 mg/kg(7,7)-P-choline was subcutaneously administrated, in a single dose, onehour prior to the formalin injection, are depicted in FIG. 2.

As can be seen in FIG. 2, the animals treated with (7,7)-P-choline spenta significant less time licking their paw compared to the non-treatedanimals. This reduction in response occurs at the chronic phase duringthe 10 to 35 minutes after the treatment with formalin.

The reduction in the animal response to formalin induced pain aftertreatment with the drug is an indication of the analgesic action of thedrug.

Example 9 Effects of BFAs Measured in the Chronic Constriction Injury(CCI) Model System (Neuropathic Pain)

The chronic constriction injury (CCI) is an animal model systemdeveloped by Bennet and Xie [(1988) Pain 33: 87-107] for producing achronic peripheral mononeuropathy in rodents.

Neuropathic pain is induced by loose ligation of the sciatic nerve andis characterized by hyperalgesia (increased sensitivity to painfulstimuli) and allodynia (the sensitivity in response to normallyinnocuous stimuli).

Male, Sprague-Dawley (SD) rats, weighing around 200-250 grams, wereanesthetized by i.p. injection of ketamine (50 mg/kg) and xylazine (10mg/kg). The common sciatic nerve was exposed at the middle of the thighof the hind right foot. Four ligatures were tied loosely around thenerve with a spacing of about 1 mm between them. The incision was closedby suturing the inner layers of the muscles with chromic gut suture, andthe outer skin with silk suture. Five to seven days later thenociceptive threshold can be evaluated by quantifying sensitivity of thefoot to mechanical stimuli or cold temperatures to assess allodynia.

The mechanical sensitivity of the animal foot is measured using the vonFrey test [Kim and Chung (1992) Pain 50: 355-63]. An automated von Freytest was employed, in which a von Frey filament is applied to theplantar surface of the animal foot and the filament is pushedautomatically at a preset rate of force/time increments into theplantar. Each measurement was repeated 2-3 times in each epsilateral andcontralateral foot of the tested animals. The time lapsed from applyingof pressure until withdrawal of the foot from the innocuous stimulus wasmeasured in the treated animals and compared to the baseline valuesobtained for each animal prior to treatment.

Results of mechanical allodynia test, measured on rats at day 15post-operation, at different time points following subcutaneousadministration of 150 mg/ml (7,7)-P-choline are illustrated in FIG. 3.As a control served CCI-operated animals injected with vehicle only.

As can be seen in FIG. 3, the treatment with (7,7)-P-cholinesignificantly. increased withdrawal threshold for at least 5 hoursfollowing administration of the drug.

The cold sensitivity is quantified by monitoring brisk foot withdrawalin response to acetone. Acetone is applied to the plantar surface of theanimal foot and the frequency of foot withdrawal is measured asdescribed by Choi et al. [(1994) Pain 59:369-376].

Another method of testing cold allodynia is by placing the animal onmetal platform immersed 1-3 mm in ice cold water (2-4° C.). The animalsare allowed to walk around on the platform for 2 minutes during whichthe numbers of foot lifting are recorded. These numbers (score) ofwithdrawals/lifting are compared to the pre-treatment scores of the sameanimals and were used to evaluate the analgesic effect of the appliedtreatment.

In another set of experiments, hyperalgesia to noxious pain isquantified by using radiated infrared (IR) heat as the pain stimulussimilarly to the procedure described above in Example 6 for the tailflick assay, except that in this case the IR is focused on the plantarhind paw of a rat. The withdrawal latency of the hind paw is recorded inanimals pre-treated with various dosages of BFAs/DP-BFAs (10-200 mg/kgbody weight) or vehicle only (control). The withdrawal latency isrecorded at 15, 45, 75, 105 and 145 minutes following s.c. injection ofthe tested drug or vehicle solution. The determined withdrawal latencyis compared to the initial withdrawal latency for each animal asmeasured at t=0 (baseline threshold), and to the withdrawal latencymeasured for the uninjured hind foot. Same behavioral tests aimed toestablish nociceptive threshold values for naive uninjured rats, areconducted on all animals one day prior to surgery.

The analgesic effect of BFAs and DP-BFAs is tested by recording increasein the withdrawal latency measured for the injured foot in the animalstreated with these drugs. The analgesic effect is also assessed by thevon Frey and cold sensitivity tests.

Example 10 Hemisection Injury of Spinal Chord (Central Neuropathic Pain)

Spinal cord hemisection is an acceptable model system for inducingcentral chronic neuropathic pain.

Male Sprague-Dawley (SD) rats weighing around 200-250 grams are deeplyanesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg). Thespinal cord is hemi-sectioned at the level of T9 to T12 with a scalpelblade without damage to the surrounding vasculature. Then the incisionis closed. Sham operated animals serve as a control group.

Behavioral tests representing mechanical and thermal allodynia asdescribed above in Example 9 are performed pre-operatively andpost-operatively for both hind limbs. The preoperative testing isperformed one day prior to surgery and serves to establish bothindividual and group baseline behaviors. The tests are performed onalternate days, starting on the fifth to seventh day following surgery,and are carried on for up to 30 days. Mechanical allodynia of the paw isquantified by measuring the number of brisk paw withdrawals in responseto normally innocuous mechanical stimuli applied by von Frey filaments[Christensen et al. (1996) Pain 68: 97-107].

The analgesic effect of the BFAs and DP-BFAs compounds is tested. Adecrease in the number of paw withdrawals in response to stimuli by vonFrey filaments is an indication for analgesia. The beneficial effectsare also assessed in the thermal tests where increase in the withdrawallatency for the ipsilateral foot of the injured animals treated with thedrugs is an indication for analgesic activity.

Conclusions: The tested branched-chain fatty acids and theirDP-derivatives are potential analgesics that may serve for treatingpain, including neuropathic pain.

The foregoing disclosure has been set forth merely to illustrate theinvention and is not intended to be limiting. Persons skilled in the artwill appreciate that many variations and modifications can be made whichdo not depart from the teaching of the present invention. Therefore, theinvention is not to be construed as restricted to the particularlydescribed embodiments, rather the scope, spirit and concept of theinvention will be more readily understood by reference to the claimswhich follow.

1. A method of treating or alleviating pain in mammals, comprisingadministering to a mammal suffering from pain, a pain-alleviating amountof a compound of the general formula:

and pharmaceutically acceptable salts thereof, wherein: R1 is asaturated or unsaturated chain of 1-18 carbons in length; R2 is asaturated or unsaturated chain of 1-18 carbons in length; and A isPO₄—X, wherein X is a hydrogen or choline.
 2. The method according toclaim 1, wherein the mammal suffering from pain is a human.
 3. Themethod according to claim 1, wherein the pain is other than chronicpain.
 4. The method according to claim 1, wherein the pain is acutepain.
 5. The method according to claim 1, wherein the pain isneuropathic pain.
 6. The method according to claim 1, wherein the painis associated with a pathological condition or disease state.
 7. Themethod according to claim 1, wherein said compound is 8-PentadecanylPhosphate.
 8. The method according to claim 1, wherein said compound is8-Pentadecanyl phosphocholine.
 9. The method according to claim 1,wherein the pain-alleviating amount of the compound is administeredbefore onset of pain.
 10. The method according to claim 1, wherein thecompound is orally, intravenously or topically administered.
 11. Themethod according to claim 1, wherein the pain is associated with apathological condition or disease state related to inflammationcondition.
 12. The method according to claim 11, wherein saidpathological condition or disease state related to inflammationcondition is selected from the group consisting of post-operative pain,toothache, pain induced by bums, muscle pain, peripheral nerve injury,headache and arthritis.